Structured Review

TaKaRa pstv28 vector
Recovery from suppression by Ppag without omega factor. Interaction of the FLAG-AtxA with the upstream region of pagA gene was compared with that of an empty vector. A <t>pSTV28</t> vector expressing no bait protein was used as a control (-). *Indicates p
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1) Product Images from "Upstream sequence-dependent suppression and AtxA-dependent activation of protective antigens in Bacillus anthracis"

Article Title: Upstream sequence-dependent suppression and AtxA-dependent activation of protective antigens in Bacillus anthracis

Journal: PeerJ

doi: 10.7717/peerj.6718

Recovery from suppression by Ppag without omega factor. Interaction of the FLAG-AtxA with the upstream region of pagA gene was compared with that of an empty vector. A pSTV28 vector expressing no bait protein was used as a control (-). *Indicates p
Figure Legend Snippet: Recovery from suppression by Ppag without omega factor. Interaction of the FLAG-AtxA with the upstream region of pagA gene was compared with that of an empty vector. A pSTV28 vector expressing no bait protein was used as a control (-). *Indicates p

Techniques Used: Plasmid Preparation, Expressing

2) Product Images from "Pannexin3 inhibits TNF‐α‐induced inflammatory response by suppressing NF‐κB signalling pathway in human dental pulp cells"

Article Title: Pannexin3 inhibits TNF‐α‐induced inflammatory response by suppressing NF‐κB signalling pathway in human dental pulp cells

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.12988

Panx3 regulates NF ‐κB signalling pathway. ( A, B ) NF ‐κB transcriptional activities in HDPC /Panx3, HDPC /Plvx, HDPC /sh RNA and HDPC /Ctrl groups were measured using dual‐luciferase reporter system. * P
Figure Legend Snippet: Panx3 regulates NF ‐κB signalling pathway. ( A, B ) NF ‐κB transcriptional activities in HDPC /Panx3, HDPC /Plvx, HDPC /sh RNA and HDPC /Ctrl groups were measured using dual‐luciferase reporter system. * P

Techniques Used: Luciferase

Panx3 regulates TNF ‐α‐induced inflammatory cytokine expression in HDPC s. qRT ‐ PCR ( A, B ) and ELISA ( C ) analysis of IL ‐1β and IL ‐6 expression in HDPC /Panx3 and HDPC /Plvx cells upon TNF ‐α stimulation for 24 hrs. qRT ‐ PCR ( D, E ) and ELISA ( F ) analysis of IL ‐1β and IL ‐6 expression in HDPC /sh RNA and HDPC /Ctrl cells upon TNF ‐α stimulation for 24 hrs. * P
Figure Legend Snippet: Panx3 regulates TNF ‐α‐induced inflammatory cytokine expression in HDPC s. qRT ‐ PCR ( A, B ) and ELISA ( C ) analysis of IL ‐1β and IL ‐6 expression in HDPC /Panx3 and HDPC /Plvx cells upon TNF ‐α stimulation for 24 hrs. qRT ‐ PCR ( D, E ) and ELISA ( F ) analysis of IL ‐1β and IL ‐6 expression in HDPC /sh RNA and HDPC /Ctrl cells upon TNF ‐α stimulation for 24 hrs. * P

Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Schematic illustration of the role of Panx3 in dental pulp inflammation. TNF ‐α could down‐regulate the expression of Panx3 via activating proteasome pathway, meanwhile the NF ‐κB might balance the effect. In addition, Panx3 could suppress NF ‐κB signalling pathway, leading to inhibition of TNF ‐α‐induced inflammatory response. The black lines indicate that the interactions have been clearly established in previous studies. The purple lines indicate the connection established in this study. MG 132, a proteasome inhibitor; BAY 11‐7082, a NF ‐κB inhibitor.
Figure Legend Snippet: Schematic illustration of the role of Panx3 in dental pulp inflammation. TNF ‐α could down‐regulate the expression of Panx3 via activating proteasome pathway, meanwhile the NF ‐κB might balance the effect. In addition, Panx3 could suppress NF ‐κB signalling pathway, leading to inhibition of TNF ‐α‐induced inflammatory response. The black lines indicate that the interactions have been clearly established in previous studies. The purple lines indicate the connection established in this study. MG 132, a proteasome inhibitor; BAY 11‐7082, a NF ‐κB inhibitor.

Techniques Used: Expressing, Inhibition

MG 132 not BAY 11‐7082 rescued the TNF ‐α‐induced down‐regulation of Panx3. Cells were pre‐treated with 2 μM BAY 11‐7082, 1 μM MG 132 or DMSO for 30 min. before TNF ‐α treatment. qRT ‐ PCR ( A, C ) and Western blot analysis ( B, D ) were then performed to determine the Panx3 expression. * P
Figure Legend Snippet: MG 132 not BAY 11‐7082 rescued the TNF ‐α‐induced down‐regulation of Panx3. Cells were pre‐treated with 2 μM BAY 11‐7082, 1 μM MG 132 or DMSO for 30 min. before TNF ‐α treatment. qRT ‐ PCR ( A, C ) and Western blot analysis ( B, D ) were then performed to determine the Panx3 expression. * P

Techniques Used: Quantitative RT-PCR, Western Blot, Expressing

TNF ‐α repressed Panx3 expression at the mRNA and protein levels in a concentration‐dependent manner in HDPC s. The mRNA and protein expression levels of Panx3 in HDPC s stimulated with TNF ‐α (0–10 ng/ml) for 24 hrs were detected by qRT ‐ PCR ( A ) and Western blot ( B ). GAPDH served as an internal control. ( C ) Immunofluorescence of Panx3 in HDPC s with or without TNF ‐α (10 ng/ml) treatment for 24 hrs. Scale bar = 200 μm. Data were expressed as mean ± SEM . * P
Figure Legend Snippet: TNF ‐α repressed Panx3 expression at the mRNA and protein levels in a concentration‐dependent manner in HDPC s. The mRNA and protein expression levels of Panx3 in HDPC s stimulated with TNF ‐α (0–10 ng/ml) for 24 hrs were detected by qRT ‐ PCR ( A ) and Western blot ( B ). GAPDH served as an internal control. ( C ) Immunofluorescence of Panx3 in HDPC s with or without TNF ‐α (10 ng/ml) treatment for 24 hrs. Scale bar = 200 μm. Data were expressed as mean ± SEM . * P

Techniques Used: Expressing, Concentration Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence

The expression of Panx3 was decreased in inflamed human ( A ) and rat dental pulpitis tissue (B). The dental pulp tissue sections were stained with HE , anti‐Panx3 antibody or anti‐ TNF ‐α antibody. HE ‐stained sections of normal and inflamed dental pulp tissue verified the progression of inflammation. Immunohistochemistry performed on dental pulp tissues showed the presence of Panx3 in normal tissue, but decreased in the inflamed pulp tissue. In contrast to Panx3 expression, TNF ‐α was increased in inflamed dental pulp tissue. Cell nuclei were visualized with haematoxylin. Scale bars are stamped in the images, respectively.
Figure Legend Snippet: The expression of Panx3 was decreased in inflamed human ( A ) and rat dental pulpitis tissue (B). The dental pulp tissue sections were stained with HE , anti‐Panx3 antibody or anti‐ TNF ‐α antibody. HE ‐stained sections of normal and inflamed dental pulp tissue verified the progression of inflammation. Immunohistochemistry performed on dental pulp tissues showed the presence of Panx3 in normal tissue, but decreased in the inflamed pulp tissue. In contrast to Panx3 expression, TNF ‐α was increased in inflamed dental pulp tissue. Cell nuclei were visualized with haematoxylin. Scale bars are stamped in the images, respectively.

Techniques Used: Expressing, Staining, Immunohistochemistry

Lentiviral‐mediated expression of Panx3 in HDPC s. ( A, B ) Cell images of HDPC s/Panx3, HDPC /Plvx, HDPC /sh RNA and HCPD /Ctrl groups were photographed in normal light (lower panels) and under a fluorescence microscope (upper panels). Protein and mRNA expression of Panx3 were determined by Western blot ( C, D ) and qRT ‐ PCR ( E, F ) analysis. * P
Figure Legend Snippet: Lentiviral‐mediated expression of Panx3 in HDPC s. ( A, B ) Cell images of HDPC s/Panx3, HDPC /Plvx, HDPC /sh RNA and HCPD /Ctrl groups were photographed in normal light (lower panels) and under a fluorescence microscope (upper panels). Protein and mRNA expression of Panx3 were determined by Western blot ( C, D ) and qRT ‐ PCR ( E, F ) analysis. * P

Techniques Used: Expressing, Fluorescence, Microscopy, Western Blot, Quantitative RT-PCR

3) Product Images from "A plant MinD homologue rescues Escherichia coli HL1 mutant (?MinDE) in the absence of MinE"

Article Title: A plant MinD homologue rescues Escherichia coli HL1 mutant (?MinDE) in the absence of MinE

Journal: BMC Microbiology

doi: 10.1186/1471-2180-9-101

Localization of AtMinD in Arabidopsis and E. coli with a GFP tag . (A to C) AtMinD-GFP transiently expressed in an Arabidopsis protoplast. Arrows denote the localization of GFP in chloroplasts. (D and G) AtMinD-GFP expressed in E. coli HL1 mutant. (E and H), GFP-AtMinD expressed in E. coli HL1 mutant. (F and I) GFP-EcMinD expressed in E. coli HL1 mutant, (J and K) GFP-EcMinC and AtMinD expressed in E. coli RC1 mutant, (L and M) GFP-EcMinC expressed in E. coli RC1 mutant, (N) Immuno blot analysis. AtMinD-GFP, GFP-AtMinD and GFP-EcMinD were expressed in the HL1 mutant; GFP-EcMinC was expressed in the RC1 mutant with AtMinD. All the cells were grown with 20 or 50 μM IPTG. (A, D, E, F, J and L), GFP; (B), Chlorophyll; (C) Overlay; (G, H, I, K and M), DIC. Bars are 5 μm.
Figure Legend Snippet: Localization of AtMinD in Arabidopsis and E. coli with a GFP tag . (A to C) AtMinD-GFP transiently expressed in an Arabidopsis protoplast. Arrows denote the localization of GFP in chloroplasts. (D and G) AtMinD-GFP expressed in E. coli HL1 mutant. (E and H), GFP-AtMinD expressed in E. coli HL1 mutant. (F and I) GFP-EcMinD expressed in E. coli HL1 mutant, (J and K) GFP-EcMinC and AtMinD expressed in E. coli RC1 mutant, (L and M) GFP-EcMinC expressed in E. coli RC1 mutant, (N) Immuno blot analysis. AtMinD-GFP, GFP-AtMinD and GFP-EcMinD were expressed in the HL1 mutant; GFP-EcMinC was expressed in the RC1 mutant with AtMinD. All the cells were grown with 20 or 50 μM IPTG. (A, D, E, F, J and L), GFP; (B), Chlorophyll; (C) Overlay; (G, H, I, K and M), DIC. Bars are 5 μm.

Techniques Used: Mutagenesis

4) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p
Figure Legend Snippet: Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p

Techniques Used: Expressing

Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).
Figure Legend Snippet: Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Standard Deviation, Software

CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.
Figure Legend Snippet: CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.

Techniques Used: Mutagenesis

Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.
Figure Legend Snippet: Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.

Techniques Used:

Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.
Figure Legend Snippet: Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.

Techniques Used: Expressing, Transgenic Assay

5) Product Images from "Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells"

Article Title: Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156819

Dose dependent inhibition of SRSF1 by Pur-alpha in glial cells. T98G cells were transfected with increasing concentrations of pEGFP-Pur-alpha plasmid (1X, 2X, and 3X) and whole cell protein extracts were prepared at 48hr post-transfection. Expression of SRSF1 and Pur-alpha were detected by Western blotting using anti-Pur-alpha and anti-SRSF1 antibodies. Grb2 was also probed in the same blots as an internal loading control. B. Signal intensities of SRSF1 expression in panel A were normalized to Grb2 levels and are shown as a bar graph. C. T98G cells were transfected with increasing concentrations of an expression plasmid encoding GFP-Pur-alpha. Total RNA was extracted and expression levels of SRSF1 mRNA transcripts were determined by Q-RT-PCR. mRNA levels of SRSF1 were normalized and are presented relative to change in relation to the control as a bar graph. All experiments were carried out in triplicate.
Figure Legend Snippet: Dose dependent inhibition of SRSF1 by Pur-alpha in glial cells. T98G cells were transfected with increasing concentrations of pEGFP-Pur-alpha plasmid (1X, 2X, and 3X) and whole cell protein extracts were prepared at 48hr post-transfection. Expression of SRSF1 and Pur-alpha were detected by Western blotting using anti-Pur-alpha and anti-SRSF1 antibodies. Grb2 was also probed in the same blots as an internal loading control. B. Signal intensities of SRSF1 expression in panel A were normalized to Grb2 levels and are shown as a bar graph. C. T98G cells were transfected with increasing concentrations of an expression plasmid encoding GFP-Pur-alpha. Total RNA was extracted and expression levels of SRSF1 mRNA transcripts were determined by Q-RT-PCR. mRNA levels of SRSF1 were normalized and are presented relative to change in relation to the control as a bar graph. All experiments were carried out in triplicate.

Techniques Used: Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

6) Product Images from "Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality"

Article Title: Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039217

The BGLF4 expression level is the key factor that determines cell fates. (A) Slide-cultured GFP-H2B-HeLa cells were transfected with DsRed-BGLF4, DsRed-K102I and DsRed-monomer. At 24 h post transfection, cells were fixed with 4% paraformaldehyde, stained for DNA using Hoechst 33258 and examined using fluorescence microscopy. (B)(C)(D) GFP-H2B-HeLa cells were seeded and transfected with (B) DsRed-monomer vector or (C)(D) Ds-Red-BGLF4. At 6 h post transfection, fields of view were selected randomly and filmed using an Axiovert 200 M inverted fluorescence microscope every 10 min.
Figure Legend Snippet: The BGLF4 expression level is the key factor that determines cell fates. (A) Slide-cultured GFP-H2B-HeLa cells were transfected with DsRed-BGLF4, DsRed-K102I and DsRed-monomer. At 24 h post transfection, cells were fixed with 4% paraformaldehyde, stained for DNA using Hoechst 33258 and examined using fluorescence microscopy. (B)(C)(D) GFP-H2B-HeLa cells were seeded and transfected with (B) DsRed-monomer vector or (C)(D) Ds-Red-BGLF4. At 6 h post transfection, fields of view were selected randomly and filmed using an Axiovert 200 M inverted fluorescence microscope every 10 min.

Techniques Used: Expressing, Cell Culture, Transfection, Staining, Fluorescence, Microscopy, Plasmid Preparation

Expression of BGLF4 elicits abnormal chromosomal structure and micronuclei formation in various cell types. (A) Slide-cultured HeLa cells with stable GFP-H2B expression were transfected with DsRed-BGLF4, DsRed-K102I or pDsRed-monomer. At 48 h or 72 h post transfection, cells were fixed, stained for DNA with Hoechst 33258 and examined using fluorescence microscopy. (B) Slide-cultured 293 T-REx B22, K9, and V4 cells were treated with 100 ng/ml doxycycline (Dox). At 72 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for DNA with Hoechst 33258. Micronucleus (MN) formation was observed using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,000 cells. Bars represent mean values in MN% (duplicates±SD). One of the two independent experiments conducted is shown. (C) Slide-cultured NPC-TW01 T-REx KIT2 and VIT7 cells were incubated with 50 ng/ml Dox for 72 h. Cells were fixed with 4% paraformaldehyde and examined using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,200-1,600 cells. An asterisk (*) indicates a statistically significant difference from vector control cells (P
Figure Legend Snippet: Expression of BGLF4 elicits abnormal chromosomal structure and micronuclei formation in various cell types. (A) Slide-cultured HeLa cells with stable GFP-H2B expression were transfected with DsRed-BGLF4, DsRed-K102I or pDsRed-monomer. At 48 h or 72 h post transfection, cells were fixed, stained for DNA with Hoechst 33258 and examined using fluorescence microscopy. (B) Slide-cultured 293 T-REx B22, K9, and V4 cells were treated with 100 ng/ml doxycycline (Dox). At 72 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for DNA with Hoechst 33258. Micronucleus (MN) formation was observed using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,000 cells. Bars represent mean values in MN% (duplicates±SD). One of the two independent experiments conducted is shown. (C) Slide-cultured NPC-TW01 T-REx KIT2 and VIT7 cells were incubated with 50 ng/ml Dox for 72 h. Cells were fixed with 4% paraformaldehyde and examined using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,200-1,600 cells. An asterisk (*) indicates a statistically significant difference from vector control cells (P

Techniques Used: Expressing, Cell Culture, Transfection, Staining, Fluorescence, Microscopy, Derivative Assay, Incubation, Plasmid Preparation

7) Product Images from "Profilin 1 Associates with Stress Granules and ALS-Linked Mutations Alter Stress Granule Dynamics"

Article Title: Profilin 1 Associates with Stress Granules and ALS-Linked Mutations Alter Stress Granule Dynamics

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.0543-14.2014

Effect of profilin 1 and ALS-linked mutations on stress granule dynamics. A , U2OS G3BP-GFP cells transiently transfected with V5-WT profilin 1 or ALS-linked mutations and treated with 0.5 m m arsenite for 30 min; B , 2 h heat shock at 43°C; and
Figure Legend Snippet: Effect of profilin 1 and ALS-linked mutations on stress granule dynamics. A , U2OS G3BP-GFP cells transiently transfected with V5-WT profilin 1 or ALS-linked mutations and treated with 0.5 m m arsenite for 30 min; B , 2 h heat shock at 43°C; and

Techniques Used: Transfection

Profilin 1 is not required for stress granule assembly or disassembly in human U2OS cells. A , U2OS-G3BP-GFP cells examined by immunofluorescence for the stress granule markers G3BP and eIF4G. Cells show stress granule assembly upon heat shock at 43°C
Figure Legend Snippet: Profilin 1 is not required for stress granule assembly or disassembly in human U2OS cells. A , U2OS-G3BP-GFP cells examined by immunofluorescence for the stress granule markers G3BP and eIF4G. Cells show stress granule assembly upon heat shock at 43°C

Techniques Used: Immunofluorescence

Profilin 1 localizes to stress granules in U2OS cells and overexpression can induce stress granule formation in untreated cells. A , U2OS G3BP-GFP cells stained for endogenous profilin 1 in untreated conditions, after 30 min. 0.5 m m arsenite, 2 h heat
Figure Legend Snippet: Profilin 1 localizes to stress granules in U2OS cells and overexpression can induce stress granule formation in untreated cells. A , U2OS G3BP-GFP cells stained for endogenous profilin 1 in untreated conditions, after 30 min. 0.5 m m arsenite, 2 h heat

Techniques Used: Over Expression, Staining

8) Product Images from "Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells"

Article Title: Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156819

SRSF1-mediated suppression of JCV T-antigen is ameliorated by Pur-alpha. A. T98G cells were transiently transfected with expression plasmids encoding JCV T-antigen, T7-tagged SRSF1 and GFP-tagged Pur-alpha as indicated. Expression of the proteins were detected by Western blotting using anti-T-antigen, anti-SRSF1, and anti-GFP antibodies. Tubulin was probed in the same blots as an internal loading control. B. Bar graph presentation T-antigen expression levels based on signal intensity of T-antigen normalized to levels of the tubulin internal loading control in panel A.
Figure Legend Snippet: SRSF1-mediated suppression of JCV T-antigen is ameliorated by Pur-alpha. A. T98G cells were transiently transfected with expression plasmids encoding JCV T-antigen, T7-tagged SRSF1 and GFP-tagged Pur-alpha as indicated. Expression of the proteins were detected by Western blotting using anti-T-antigen, anti-SRSF1, and anti-GFP antibodies. Tubulin was probed in the same blots as an internal loading control. B. Bar graph presentation T-antigen expression levels based on signal intensity of T-antigen normalized to levels of the tubulin internal loading control in panel A.

Techniques Used: Transfection, Expressing, Western Blot

Pur-alpha blocks SRSF1 induced inhibition of JCV DNA replication. A. DpnI replication assay in PHFA cells transfected with replication competent JCV plasmid DNA and expression plasmids encoding JCV T-antigen, T7-tagged SRSF1, and GFP-tagged Pur-alpha as indicated. DNA and protein lysates were prepared from cells harvested 7 days post-transfection. DpnI digestion of DNA was performed to distinguish newly replicated DNA from the transfected input plasmid DNA. B. Results from DpnI assay in Panel A are presented as a bar graph. C. Western blot analysis of protein lysates harvested in parallel to the DpnI assay in Panel A for the expression of T7-tagged SRSF1 and GFP-tagged Pur-alpha. Tubulin was probed in the same blots as loading control.
Figure Legend Snippet: Pur-alpha blocks SRSF1 induced inhibition of JCV DNA replication. A. DpnI replication assay in PHFA cells transfected with replication competent JCV plasmid DNA and expression plasmids encoding JCV T-antigen, T7-tagged SRSF1, and GFP-tagged Pur-alpha as indicated. DNA and protein lysates were prepared from cells harvested 7 days post-transfection. DpnI digestion of DNA was performed to distinguish newly replicated DNA from the transfected input plasmid DNA. B. Results from DpnI assay in Panel A are presented as a bar graph. C. Western blot analysis of protein lysates harvested in parallel to the DpnI assay in Panel A for the expression of T7-tagged SRSF1 and GFP-tagged Pur-alpha. Tubulin was probed in the same blots as loading control.

Techniques Used: Inhibition, Transfection, Plasmid Preparation, Expressing, Western Blot

9) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p
Figure Legend Snippet: Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p

Techniques Used: Expressing

Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).
Figure Legend Snippet: Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Standard Deviation, Software

CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.
Figure Legend Snippet: CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.

Techniques Used: Mutagenesis

Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.
Figure Legend Snippet: Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.

Techniques Used:

Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.
Figure Legend Snippet: Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.

Techniques Used: Expressing, Transgenic Assay

10) Product Images from "Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells"

Article Title: Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells

Journal: Scientific Reports

doi: 10.1038/s41598-018-24953-4

eGFP-Affimers 6, 14 and 24 label F-actin. ( A ) SDS PAGE gels of protein samples (to show approximately equivalent loading of samples) from transfected cells as indicated, and associated western blot ( B ) using an antibody for eGFP. ( C ) Fluorescent images of fixed Hela cells expressing fluorescent eGFP-Affimer constructs (green in merged image), co-stained with fluorescent phalloidin (magenta in merged image). Images were captured using the Deltavision deconvolution microscope. Arrows for Affimer 6 and 14 show weak staining of thin protrusions by the eGFP-Affimer compared to phalloidin. Arrows for Affimer 24 show the accumulation of Affimer 24 in focal adhesions. Scale bar as shown.
Figure Legend Snippet: eGFP-Affimers 6, 14 and 24 label F-actin. ( A ) SDS PAGE gels of protein samples (to show approximately equivalent loading of samples) from transfected cells as indicated, and associated western blot ( B ) using an antibody for eGFP. ( C ) Fluorescent images of fixed Hela cells expressing fluorescent eGFP-Affimer constructs (green in merged image), co-stained with fluorescent phalloidin (magenta in merged image). Images were captured using the Deltavision deconvolution microscope. Arrows for Affimer 6 and 14 show weak staining of thin protrusions by the eGFP-Affimer compared to phalloidin. Arrows for Affimer 24 show the accumulation of Affimer 24 in focal adhesions. Scale bar as shown.

Techniques Used: SDS Page, Transfection, Western Blot, Expressing, Construct, Staining, Microscopy

Binding of Affimers to F-actin in vitro . ( A ) Shows example gels from one of the experiments with Affimer 24 in which increasing concentrations of Affimer were mixed with 2.5μM F-actin. Lanes show supernatant (S) and pellet (P) from sedimentation of the mixtures. ( B ) Shows the mean values from three separate experiments, together with the standard error. Dashed lines indicate the quadratic fits to the data for Affimers 6, 14 and 24. The dashed line for Affimer 2 is a linear fit.
Figure Legend Snippet: Binding of Affimers to F-actin in vitro . ( A ) Shows example gels from one of the experiments with Affimer 24 in which increasing concentrations of Affimer were mixed with 2.5μM F-actin. Lanes show supernatant (S) and pellet (P) from sedimentation of the mixtures. ( B ) Shows the mean values from three separate experiments, together with the standard error. Dashed lines indicate the quadratic fits to the data for Affimers 6, 14 and 24. The dashed line for Affimer 2 is a linear fit.

Techniques Used: Binding Assay, In Vitro, Sedimentation

Testing the ability of purified Affimer to stain the actin cytoskeleton in paraformaldehyde fixed cells. ( A ) Hela cells were stained with each of the 4 different Affimers, using Affimers biotinylated on the C-terminal cysteine and visualized with Alexa 546 streptavidin (shown in magenta in merged image). Cells were co-stained with Alexa 488 phalloidin (shown in green in merged images). Scale bar as shown. ( B ) Sf 9 cells were fixed and stained with Affimer 14, using a direct dye label (Alexa 488) on the C-terminal cysteine (shown in magenta) and co-stained with Alexa 546 phalloidin (shown in green in the merged image). Scale bar as shown.
Figure Legend Snippet: Testing the ability of purified Affimer to stain the actin cytoskeleton in paraformaldehyde fixed cells. ( A ) Hela cells were stained with each of the 4 different Affimers, using Affimers biotinylated on the C-terminal cysteine and visualized with Alexa 546 streptavidin (shown in magenta in merged image). Cells were co-stained with Alexa 488 phalloidin (shown in green in merged images). Scale bar as shown. ( B ) Sf 9 cells were fixed and stained with Affimer 14, using a direct dye label (Alexa 488) on the C-terminal cysteine (shown in magenta) and co-stained with Alexa 546 phalloidin (shown in green in the merged image). Scale bar as shown.

Techniques Used: Purification, Staining

). eGFP-Affimer 6 was not recruited into these regions, but was co-localised with mCherry-actin on stable filopodial structures (arrowed) and stress-fiber like structures (arrowheads).
Figure Legend Snippet: ). eGFP-Affimer 6 was not recruited into these regions, but was co-localised with mCherry-actin on stable filopodial structures (arrowed) and stress-fiber like structures (arrowheads).

Techniques Used:

FRAP demonstrates that the eGFP-Affimers exchange onto F-actin at different rates ( A ). Note that the example frames post-bleach for Lifeact are separated by much shorter intervals than for the remaining constructs, as fluorescence recovery was very fast for this construct. Images captured using the LSM 880 confocal. ( B ) example raw traces (corrected for background and any photobleaching) for the images shown in A, fit using a single exponential curve (superimposed as a red line). The data were fit using using Graph Pad Prizm. Scale bar (2 μm) applies to all images. ( C ) Show estimates of the half times for fluorescence recovery, from the exponential fits, for each of the Affimers, F-tractin and Lifeact. The mean +/− SEM is shown as bars superimposed onto the plots individual values. (N = 18, 8, 19, 17, 20 for Lifeact, F-tractin, Affimer 6, Affimer 14 and Affimer 24, respectively). Levels of significance between measurements are indicated on the graph. *P
Figure Legend Snippet: FRAP demonstrates that the eGFP-Affimers exchange onto F-actin at different rates ( A ). Note that the example frames post-bleach for Lifeact are separated by much shorter intervals than for the remaining constructs, as fluorescence recovery was very fast for this construct. Images captured using the LSM 880 confocal. ( B ) example raw traces (corrected for background and any photobleaching) for the images shown in A, fit using a single exponential curve (superimposed as a red line). The data were fit using using Graph Pad Prizm. Scale bar (2 μm) applies to all images. ( C ) Show estimates of the half times for fluorescence recovery, from the exponential fits, for each of the Affimers, F-tractin and Lifeact. The mean +/− SEM is shown as bars superimposed onto the plots individual values. (N = 18, 8, 19, 17, 20 for Lifeact, F-tractin, Affimer 6, Affimer 14 and Affimer 24, respectively). Levels of significance between measurements are indicated on the graph. *P

Techniques Used: Construct, Fluorescence

Testing the ability of purified Affimer to stain the actin cytoskeleton in methanol fixed cells. Hela cells were stained with each of the 4 different Affimers, using Affimers biotinylated on the C-terminal cysteine and visualized with Atto 488 streptavidin (shown in green in merged image). Cells were co-stained with an antibody to actin (AC-40), visualised with anti-mouse Alexa 546 antibody (shown in magenta in merged image). Co-localisation of magenta and green results in white. Scale bar as shown.
Figure Legend Snippet: Testing the ability of purified Affimer to stain the actin cytoskeleton in methanol fixed cells. Hela cells were stained with each of the 4 different Affimers, using Affimers biotinylated on the C-terminal cysteine and visualized with Atto 488 streptavidin (shown in green in merged image). Cells were co-stained with an antibody to actin (AC-40), visualised with anti-mouse Alexa 546 antibody (shown in magenta in merged image). Co-localisation of magenta and green results in white. Scale bar as shown.

Techniques Used: Purification, Staining

FRAP demonstrates that Affimers do not affect turnover dynamics of the actin cytoskeleton. ( A ). The red circle denotes the photobleached area. Scale bar (2 μm) as shown. ( B ) An example FRAP recovery curve for mCherry actin fluorescence recovery fit with a single exponential (red line) and double exponential (blue line). Fitted lines are almost identical. ( C ) Shows the values for τ measured from single exponential fits of mCherry-actin fluorescence recovery, bleached in the presence of eGFP-Lifeact, eGFP-Affimer 6, eGFP-Affimer 14 or eGFP-Affimer 24, or in their absence. T-tests were performed to determine any significant differences. None of the values were significantly different (N = 10 (mCherry-actin alone), and N = 10, 13, 10, and 8 for mCherry-actin recovery in cells co-transfected with Lifeact, Affimer 6, Affimer 14, and Affimer 24 respectively).
Figure Legend Snippet: FRAP demonstrates that Affimers do not affect turnover dynamics of the actin cytoskeleton. ( A ). The red circle denotes the photobleached area. Scale bar (2 μm) as shown. ( B ) An example FRAP recovery curve for mCherry actin fluorescence recovery fit with a single exponential (red line) and double exponential (blue line). Fitted lines are almost identical. ( C ) Shows the values for τ measured from single exponential fits of mCherry-actin fluorescence recovery, bleached in the presence of eGFP-Lifeact, eGFP-Affimer 6, eGFP-Affimer 14 or eGFP-Affimer 24, or in their absence. T-tests were performed to determine any significant differences. None of the values were significantly different (N = 10 (mCherry-actin alone), and N = 10, 13, 10, and 8 for mCherry-actin recovery in cells co-transfected with Lifeact, Affimer 6, Affimer 14, and Affimer 24 respectively).

Techniques Used: Fluorescence, Transfection

Competition of Affimers with myosin and each other for binding to F-actin. ( A ) Competition assay for combinations of 2 Affimers. The values shown are the mean of three separate experiments together with the standard deviation. ( B ) 2.5 µM Myo5b S1 (subfragment 1) co-sediments with 2.5 µM actin in the absence of ATP. Lanes show Myo5b S1 (supernatant (S) and pellet (P)), actin (S and P) and the combination of Myo5b S1 and actin. ( C ) An example SDS PAGE gel from a competition experiment between 2 μM Myo5b S1 and 0–3 μM Affimer 14 for binding to 2 μM F-actin. HC indicates the position of the heavy chain of Myo5b S1. ( D ) Results for the competition assay between Myo5b S1 and Affimer 2. ( E ) Competition assay results for Myo5b S1 and Affimer 14. ( F ) Effects of increasing Affimer 14 concentration on Myo5b S1 ATPase activated by 4 μM F-actin.
Figure Legend Snippet: Competition of Affimers with myosin and each other for binding to F-actin. ( A ) Competition assay for combinations of 2 Affimers. The values shown are the mean of three separate experiments together with the standard deviation. ( B ) 2.5 µM Myo5b S1 (subfragment 1) co-sediments with 2.5 µM actin in the absence of ATP. Lanes show Myo5b S1 (supernatant (S) and pellet (P)), actin (S and P) and the combination of Myo5b S1 and actin. ( C ) An example SDS PAGE gel from a competition experiment between 2 μM Myo5b S1 and 0–3 μM Affimer 14 for binding to 2 μM F-actin. HC indicates the position of the heavy chain of Myo5b S1. ( D ) Results for the competition assay between Myo5b S1 and Affimer 2. ( E ) Competition assay results for Myo5b S1 and Affimer 14. ( F ) Effects of increasing Affimer 14 concentration on Myo5b S1 ATPase activated by 4 μM F-actin.

Techniques Used: Binding Assay, Competitive Binding Assay, Standard Deviation, SDS Page, Concentration Assay

Screening of the Affimer clones for actin binding using phage ELISA. 24 clones were tested in wells coated with actin, and in wells containing only buffer. Absorbance of oxidised TMB at 620 nm was recorded. Clones were sequenced and those with unique sequences, and with the highest level of signal above background (clones 2, 6, 14 and 24) were selected for further analysis. All other clones that showed affinity for actin were identical to one or other of the selected clones.
Figure Legend Snippet: Screening of the Affimer clones for actin binding using phage ELISA. 24 clones were tested in wells coated with actin, and in wells containing only buffer. Absorbance of oxidised TMB at 620 nm was recorded. Clones were sequenced and those with unique sequences, and with the highest level of signal above background (clones 2, 6, 14 and 24) were selected for further analysis. All other clones that showed affinity for actin were identical to one or other of the selected clones.

Techniques Used: Clone Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Stills from time-lapse movies for the eGFP-Affimers, F-tractin and Lifeact in live B16 cells. Time shown is in seconds. Arrows show small thin protrusions (for F-tractin and Lifeact). Arrows in panels for Affimer 6 indicate cellular regions with dynamic actin filament behaviour. Arrows in panels for Affimer 14 indicate a motile single filament bundle. Arrows for Affimer 24 indicate accumulation of short actin filaments at the base of the lamella at 110 and 170 sec. Images captured using the Zeiss 880 LSM confocal using the Airyscan. Scale bar (2 μm) applies for all images.
Figure Legend Snippet: Stills from time-lapse movies for the eGFP-Affimers, F-tractin and Lifeact in live B16 cells. Time shown is in seconds. Arrows show small thin protrusions (for F-tractin and Lifeact). Arrows in panels for Affimer 6 indicate cellular regions with dynamic actin filament behaviour. Arrows in panels for Affimer 14 indicate a motile single filament bundle. Arrows for Affimer 24 indicate accumulation of short actin filaments at the base of the lamella at 110 and 170 sec. Images captured using the Zeiss 880 LSM confocal using the Airyscan. Scale bar (2 μm) applies for all images.

Techniques Used: Size-exclusion Chromatography

11) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p
Figure Legend Snippet: Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p

Techniques Used: Expressing

Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).
Figure Legend Snippet: Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Standard Deviation, Software

CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.
Figure Legend Snippet: CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.

Techniques Used: Mutagenesis

Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.
Figure Legend Snippet: Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.

Techniques Used:

Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.
Figure Legend Snippet: Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.

Techniques Used: Expressing, Transgenic Assay

12) Product Images from "A plant MinD homologue rescues Escherichia coli HL1 mutant (?MinDE) in the absence of MinE"

Article Title: A plant MinD homologue rescues Escherichia coli HL1 mutant (?MinDE) in the absence of MinE

Journal: BMC Microbiology

doi: 10.1186/1471-2180-9-101

Localization of AtMinD in Arabidopsis and E. coli with a GFP tag . (A to C) AtMinD-GFP transiently expressed in an Arabidopsis protoplast. Arrows denote the localization of GFP in chloroplasts. (D and G) AtMinD-GFP expressed in E. coli HL1 mutant. (E and H), GFP-AtMinD expressed in E. coli HL1 mutant. (F and I) GFP-EcMinD expressed in E. coli HL1 mutant, (J and K) GFP-EcMinC and AtMinD expressed in E. coli RC1 mutant, (L and M) GFP-EcMinC expressed in E. coli RC1 mutant, (N) Immuno blot analysis. AtMinD-GFP, GFP-AtMinD and GFP-EcMinD were expressed in the HL1 mutant; GFP-EcMinC was expressed in the RC1 mutant with AtMinD. All the cells were grown with 20 or 50 μM IPTG. (A, D, E, F, J and L), GFP; (B), Chlorophyll; (C) Overlay; (G, H, I, K and M), DIC. Bars are 5 μm.
Figure Legend Snippet: Localization of AtMinD in Arabidopsis and E. coli with a GFP tag . (A to C) AtMinD-GFP transiently expressed in an Arabidopsis protoplast. Arrows denote the localization of GFP in chloroplasts. (D and G) AtMinD-GFP expressed in E. coli HL1 mutant. (E and H), GFP-AtMinD expressed in E. coli HL1 mutant. (F and I) GFP-EcMinD expressed in E. coli HL1 mutant, (J and K) GFP-EcMinC and AtMinD expressed in E. coli RC1 mutant, (L and M) GFP-EcMinC expressed in E. coli RC1 mutant, (N) Immuno blot analysis. AtMinD-GFP, GFP-AtMinD and GFP-EcMinD were expressed in the HL1 mutant; GFP-EcMinC was expressed in the RC1 mutant with AtMinD. All the cells were grown with 20 or 50 μM IPTG. (A, D, E, F, J and L), GFP; (B), Chlorophyll; (C) Overlay; (G, H, I, K and M), DIC. Bars are 5 μm.

Techniques Used: Mutagenesis

13) Product Images from "The lncRNA RMEL3 protects immortalized cells from serum withdrawal-induced growth arrest and promotes melanoma cell proliferation and tumor growth"

Article Title: The lncRNA RMEL3 protects immortalized cells from serum withdrawal-induced growth arrest and promotes melanoma cell proliferation and tumor growth

Journal: Pigment cell & melanoma research

doi: 10.1111/pcmr.12751

RMEL3 lncRNA levels decrease in BRAFV600E mutant melanoma cells after treatment with BRAF and MEK inhibitors. (A - D) RT-qPCR expression analyses of (A, B) RMEL3 lncRNA and (C, D) FOXD3 (positive control) in melanoma cell lines treated with the BRAF inhibitor vemurafenib (PLX4032) (1 μM, 6 h or 10 μM, 48 h) and respective controls (vehicle), as indicated. (E) Western blot analysis for phosphorylated ERK (pERK) and total ERK (tERK) in total protein lysates from melanoma cells treated with BRAF inhibitor (1 μM, 6 h or 10 μM, 48 h) or vehicle. (F) RT-qPCR expression analysis of RMEL3 in UACC-62 melanoma cell line treated with the MEK inhibitor PD98059 (25 μM) or vehicle for 48 hr, and corresponding Western blot analysis for phosphorylated ERK (pERK) and total ERK (tERK) in total protein lysates of treated and control melanoma cells. For all RT-qPCR analyses, relative expression was calculated according to 2 −ΔΔCT method using TBP (Tata-box binding protein) as endogenous control and the normalized Ct of the sample treated with the vehicle alone as reference. Error bars represent SEM of 3 independent experiments. *p
Figure Legend Snippet: RMEL3 lncRNA levels decrease in BRAFV600E mutant melanoma cells after treatment with BRAF and MEK inhibitors. (A - D) RT-qPCR expression analyses of (A, B) RMEL3 lncRNA and (C, D) FOXD3 (positive control) in melanoma cell lines treated with the BRAF inhibitor vemurafenib (PLX4032) (1 μM, 6 h or 10 μM, 48 h) and respective controls (vehicle), as indicated. (E) Western blot analysis for phosphorylated ERK (pERK) and total ERK (tERK) in total protein lysates from melanoma cells treated with BRAF inhibitor (1 μM, 6 h or 10 μM, 48 h) or vehicle. (F) RT-qPCR expression analysis of RMEL3 in UACC-62 melanoma cell line treated with the MEK inhibitor PD98059 (25 μM) or vehicle for 48 hr, and corresponding Western blot analysis for phosphorylated ERK (pERK) and total ERK (tERK) in total protein lysates of treated and control melanoma cells. For all RT-qPCR analyses, relative expression was calculated according to 2 −ΔΔCT method using TBP (Tata-box binding protein) as endogenous control and the normalized Ct of the sample treated with the vehicle alone as reference. Error bars represent SEM of 3 independent experiments. *p

Techniques Used: Mutagenesis, Quantitative RT-PCR, Expressing, Positive Control, Western Blot, Binding Assay

High RMEL3 expression predominates in BRAFV600E followed by NRAS mutant melanoma cell lines, as well as in nodular/ superficial spreading in comparison with acral/mucosal melanomas. (A) RT-qPCR analysis of RMEL3 RNA levels in melanocytes and melanoma cell lines of indicated genotype regarding major câncer drivers (BRAF, NRAS, and NF1). (B) RT-qPCR analysis of RMEL3 expression in melanoma subtypes: acral/mucosal and nodular/superficial spreading cutaneous melanoma, for which patient specimens were obtained from the Barretos Cancer Hospital, São Paulo state, Brazil. In A and B, relative expression was calculated according to 2 −ΔΔCT method usingTBP (Tata-box binding protein) as endogenous control, and the mean levels of RMEL3 RNA in different foreskin human melanocyte primary cultures were used as reference in both A and B, totaling the use of four different melanocyte cultures derived from different donors. Error bars represent SEM of 3 independent experiments. ***p
Figure Legend Snippet: High RMEL3 expression predominates in BRAFV600E followed by NRAS mutant melanoma cell lines, as well as in nodular/ superficial spreading in comparison with acral/mucosal melanomas. (A) RT-qPCR analysis of RMEL3 RNA levels in melanocytes and melanoma cell lines of indicated genotype regarding major câncer drivers (BRAF, NRAS, and NF1). (B) RT-qPCR analysis of RMEL3 expression in melanoma subtypes: acral/mucosal and nodular/superficial spreading cutaneous melanoma, for which patient specimens were obtained from the Barretos Cancer Hospital, São Paulo state, Brazil. In A and B, relative expression was calculated according to 2 −ΔΔCT method usingTBP (Tata-box binding protein) as endogenous control, and the mean levels of RMEL3 RNA in different foreskin human melanocyte primary cultures were used as reference in both A and B, totaling the use of four different melanocyte cultures derived from different donors. Error bars represent SEM of 3 independent experiments. ***p

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Binding Assay, Derivative Assay

Ectopic expression of human RMEL3 protects NIH3T3 murine fibroblasts from serum withdrawal-induced growth arrest/ apoptosis. (A) Schematic representation of RMEL3 locus (above), its transcript (center), and the region cloned into expression vectors (below). (B) Efficiency of exogenous RMEL3 induction in cells transduced with pLVX-RMEL3 or pLVX-TP (as control) after treatment with doxycycline (1 μM, 24 h), analyzed by RT-qPCR. ND (not-detected). Relative expression was calculated according to ΔCT using TBP (Tatabox binding protein) as endogenous control. (C-E) Functional assays using NIH3T3 cells stably transduced with pLVX-RMEL3 or pLVX-TP (as control) in the presence of doxycycline (1 μM). (C) Proliferation rates. During the time course of the assay, cells were maintained under serum starvation (in medium supplemented with 0.5% FBS). After the indicated time points, cells were stained with crystal violet and cell density was quantified according to the absorbance in an ELISA microplate reader. ***p
Figure Legend Snippet: Ectopic expression of human RMEL3 protects NIH3T3 murine fibroblasts from serum withdrawal-induced growth arrest/ apoptosis. (A) Schematic representation of RMEL3 locus (above), its transcript (center), and the region cloned into expression vectors (below). (B) Efficiency of exogenous RMEL3 induction in cells transduced with pLVX-RMEL3 or pLVX-TP (as control) after treatment with doxycycline (1 μM, 24 h), analyzed by RT-qPCR. ND (not-detected). Relative expression was calculated according to ΔCT using TBP (Tatabox binding protein) as endogenous control. (C-E) Functional assays using NIH3T3 cells stably transduced with pLVX-RMEL3 or pLVX-TP (as control) in the presence of doxycycline (1 μM). (C) Proliferation rates. During the time course of the assay, cells were maintained under serum starvation (in medium supplemented with 0.5% FBS). After the indicated time points, cells were stained with crystal violet and cell density was quantified according to the absorbance in an ELISA microplate reader. ***p

Techniques Used: Expressing, Clone Assay, Transduction, Quantitative RT-PCR, Binding Assay, Functional Assay, Stable Transfection, Staining, Enzyme-linked Immunosorbent Assay

Overexpression of RMEL3 increases proliferation of a BRAFV600E mutant, RMEL3-low expresser, melanoma cell line. (A) Efficiency of stable expression of RMEL3 RNA in VM10 melanoma cells transduced with pLJM1-RMEL3, analyzed by RT-qPCR. Relative expression was calculated according to 2 −ΔΔCT method using TBP (Tata-box binding protein) as endogenous control and the normalized Ct of cells transduced with control vector (pLJM1-EGFP) as reference. (B) Proliferation of VM10 cells transduced with pLJM1-RMEL3 or pLJM1-EGFP as control. Cells were cultured in medium supplemented with 10% FBS, and after the indicated time points, they were stained with crystal violet for quantification of cell density according to the absorbance in an ELISA microplate plate reader. Error bars represent SEM of 3 independent experiments. Asterisks indicate statistically significant differences between groups based on unpaired parametric Student’s t test. Day 2 (**p
Figure Legend Snippet: Overexpression of RMEL3 increases proliferation of a BRAFV600E mutant, RMEL3-low expresser, melanoma cell line. (A) Efficiency of stable expression of RMEL3 RNA in VM10 melanoma cells transduced with pLJM1-RMEL3, analyzed by RT-qPCR. Relative expression was calculated according to 2 −ΔΔCT method using TBP (Tata-box binding protein) as endogenous control and the normalized Ct of cells transduced with control vector (pLJM1-EGFP) as reference. (B) Proliferation of VM10 cells transduced with pLJM1-RMEL3 or pLJM1-EGFP as control. Cells were cultured in medium supplemented with 10% FBS, and after the indicated time points, they were stained with crystal violet for quantification of cell density according to the absorbance in an ELISA microplate plate reader. Error bars represent SEM of 3 independent experiments. Asterisks indicate statistically significant differences between groups based on unpaired parametric Student’s t test. Day 2 (**p

Techniques Used: Over Expression, Mutagenesis, Expressing, Transduction, Quantitative RT-PCR, Binding Assay, Plasmid Preparation, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

14) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

15) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

16) Product Images from "Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression"

Article Title: Impact of Adenovirus E4-ORF3 Oligomerization and Protein Localization on Cellular Gene Expression

Journal: Viruses

doi: 10.3390/v7052428

Validation of select E4-ORF3 targets from ( A ) cluster 1 or ( B ) cluster 2 by RT-qPCR with relative quantification. Bars represent Log 2 fold expression change in dox-treated Tet-E4-ORF3 or U2OS-Tet cells relative to mock treated Tet-E4-ORF3 or U2OS-Tet cells, normalized to GAPDH at 6 and 24 hours post-induction. Error bars = S.D., n = 3.
Figure Legend Snippet: Validation of select E4-ORF3 targets from ( A ) cluster 1 or ( B ) cluster 2 by RT-qPCR with relative quantification. Bars represent Log 2 fold expression change in dox-treated Tet-E4-ORF3 or U2OS-Tet cells relative to mock treated Tet-E4-ORF3 or U2OS-Tet cells, normalized to GAPDH at 6 and 24 hours post-induction. Error bars = S.D., n = 3.

Techniques Used: Quantitative RT-PCR, Expressing

17) Product Images from "The receptor genes PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in the pearl oyster Pinctada fucata"

Article Title: The receptor genes PfBMPR1B and PfBAMBI are involved in regulating shell biomineralization in the pearl oyster Pinctada fucata

Journal: Scientific Reports

doi: 10.1038/s41598-017-10011-y

Interactions among Bambi, Bmpr1b and members of the TGFβ/BMP signaling pathway in Pinctada fucata detected by a yeast two-hybrid system. The plasmids are cotransformed into the yeast AH109 and spread on the dropout synthetically defined (SD) medium. The white colonies on SD-Leu-Trp medium (SD-2D) indicate that the plasmids are successfully transformed. The blue colonies on SD-Ade-His-Leu-Trp/X-α-Gal medium (SD-4D- X-α-Gal) indicate the presence of an interaction between two proteins, comparable with that of the positive (AD-T + BD-p53), negative (AD-T + BD-Lam) and empty plasmid (AD + BD) controls. The triangle indicates that the yeast is gradient diluted five times with sterilized water before spreading on the medium.
Figure Legend Snippet: Interactions among Bambi, Bmpr1b and members of the TGFβ/BMP signaling pathway in Pinctada fucata detected by a yeast two-hybrid system. The plasmids are cotransformed into the yeast AH109 and spread on the dropout synthetically defined (SD) medium. The white colonies on SD-Leu-Trp medium (SD-2D) indicate that the plasmids are successfully transformed. The blue colonies on SD-Ade-His-Leu-Trp/X-α-Gal medium (SD-4D- X-α-Gal) indicate the presence of an interaction between two proteins, comparable with that of the positive (AD-T + BD-p53), negative (AD-T + BD-Lam) and empty plasmid (AD + BD) controls. The triangle indicates that the yeast is gradient diluted five times with sterilized water before spreading on the medium.

Techniques Used: Transformation Assay, Laser Capture Microdissection, Plasmid Preparation

18) Product Images from "The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer *"

Article Title: The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.110296

HspL co-fractionated with VirB complexes and physically interacted with VirB8. A , membrane fractions of A. tumefaciens wild type strain NT1RE(pJK270) grown in I-medium in the presence of AS at 25 °C for 16 h were subjected for 2% DDM extraction
Figure Legend Snippet: HspL co-fractionated with VirB complexes and physically interacted with VirB8. A , membrane fractions of A. tumefaciens wild type strain NT1RE(pJK270) grown in I-medium in the presence of AS at 25 °C for 16 h were subjected for 2% DDM extraction

Techniques Used:

Chaperone activity assays of HspL and its variants (HspLF98A and HspLG118A) using GST-VirB8 as substrate. Thermal aggregation protection assays using GST-VirB8 (600 n m ) was carried out at 50 °C in the absence (○) or presence of HspL-His
Figure Legend Snippet: Chaperone activity assays of HspL and its variants (HspLF98A and HspLG118A) using GST-VirB8 as substrate. Thermal aggregation protection assays using GST-VirB8 (600 n m ) was carried out at 50 °C in the absence (○) or presence of HspL-His

Techniques Used: Activity Assay

19) Product Images from "Aquareovirus NS80 Recruits Viral Proteins to Its Inclusions, and Its C-Terminal Domain Is the Primary Driving Force for Viral Inclusion Formation"

Article Title: Aquareovirus NS80 Recruits Viral Proteins to Its Inclusions, and Its C-Terminal Domain Is the Primary Driving Force for Viral Inclusion Formation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0055334

Colocalization, Western Blot and co-IP of NS80 and NS38 in transfected cells. A . IF microscopy of single transfection with pCI-neo-NS38 (up row) and cotransfection with pCI-neo-NS80 and pCI-neo-NS38 at 6 h, 12 h, 18 h p.t. The subcellular localization of NS80 was visualized by immunostaining with rabbit anti-NS80 serum followed by FITC-conjugated goat anti-rabbit IgG (green), NS38 was detected by immunostaining with mouse anti-NS38 polyclonal antibody followed by Texas Red-conjugated goat anti-mouse IgG (red). Nuclei were counterstained with DAPI (blue). B . IF microscopy of Vero cells cotransfected with pEGFP-C1-NS80 and pCI-neo-NS38 (top row) or pEGFP-C1-NS38 and pCI-neo-NS80 (bottom row). Cells were immunostained with corresponding antibodies. C . IB analysis of NS80 or NS38 in Vero cells. Cells were singly transfected or cotransfected with plasmids pCI-neo-NS80 and pCI-neo-NS38. Transfected cells were collected at 20 h p.t., lysed and analyzed by immunoblotting. D . Co-IP assay of NS80 and NS38 in transfected Vero cells. The transfected cells were harvested at 20 h p.t., lysed and immunoprecipitated with NS38-specific rabbit antiserum (upper panel) or NS80-specific mouse antiserum (lower panel), immunoprecipitated proteins were analyzed using IB analysis as indicated in Fig. 4C .
Figure Legend Snippet: Colocalization, Western Blot and co-IP of NS80 and NS38 in transfected cells. A . IF microscopy of single transfection with pCI-neo-NS38 (up row) and cotransfection with pCI-neo-NS80 and pCI-neo-NS38 at 6 h, 12 h, 18 h p.t. The subcellular localization of NS80 was visualized by immunostaining with rabbit anti-NS80 serum followed by FITC-conjugated goat anti-rabbit IgG (green), NS38 was detected by immunostaining with mouse anti-NS38 polyclonal antibody followed by Texas Red-conjugated goat anti-mouse IgG (red). Nuclei were counterstained with DAPI (blue). B . IF microscopy of Vero cells cotransfected with pEGFP-C1-NS80 and pCI-neo-NS38 (top row) or pEGFP-C1-NS38 and pCI-neo-NS80 (bottom row). Cells were immunostained with corresponding antibodies. C . IB analysis of NS80 or NS38 in Vero cells. Cells were singly transfected or cotransfected with plasmids pCI-neo-NS80 and pCI-neo-NS38. Transfected cells were collected at 20 h p.t., lysed and analyzed by immunoblotting. D . Co-IP assay of NS80 and NS38 in transfected Vero cells. The transfected cells were harvested at 20 h p.t., lysed and immunoprecipitated with NS38-specific rabbit antiserum (upper panel) or NS80-specific mouse antiserum (lower panel), immunoprecipitated proteins were analyzed using IB analysis as indicated in Fig. 4C .

Techniques Used: Western Blot, Co-Immunoprecipitation Assay, Transfection, Microscopy, Cotransfection, Immunostaining, Immunoprecipitation

Colocalization of NS80 and GFP-VP4 in inclusion structures. A . Fluorescence microscope or IF analysis of Vero cells transfected with plasmid pEGFP-C1-VP4 (row1) or cotransfected with pEGFP-C1 and pCI-neo-NS80 (row2). B . IF analysis of Vero cells cotransfected with pEGFP-C1-VP4 and pCI-neo-NS80 (row 1) or pEGFP-C1-VP4 and pCI-neo-NS38 (row 2). Transfected cells were immunostained with corresponding antibodies. Nuclei were stained blue with DAPI.
Figure Legend Snippet: Colocalization of NS80 and GFP-VP4 in inclusion structures. A . Fluorescence microscope or IF analysis of Vero cells transfected with plasmid pEGFP-C1-VP4 (row1) or cotransfected with pEGFP-C1 and pCI-neo-NS80 (row2). B . IF analysis of Vero cells cotransfected with pEGFP-C1-VP4 and pCI-neo-NS80 (row 1) or pEGFP-C1-VP4 and pCI-neo-NS38 (row 2). Transfected cells were immunostained with corresponding antibodies. Nuclei were stained blue with DAPI.

Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Staining

20) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

21) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

22) Product Images from "A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes"

Article Title: A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB–bHLH–WD40 Ternary Complexes

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061686

( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.
Figure Legend Snippet: ( A ) Relative expression of flavonoid biosynthetic pathway genes in CsMYB5e overexpression tobacco petals. ( B ) Relative expression of flavonoid biosynthetic pathway genes in CsWD40 and CsMYB5e overexpressing tobacco petals. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase.

Techniques Used: Expressing, Over Expression

Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.
Figure Legend Snippet: Anthocyanin and proanthocyanidin accumulation variated in petals of co-overexpressing CsWD40 and CsMYB5e transgenic tobacco. ( A ) The relative content of anthocyanin was calculated on the record of absorbance at 530 nm. ( B ) The relative content of proanthocyanidin was calculated on the record of absorbance at 640 nm after reaction with DMACA regent. ( C ) RT–PCR determination of the CsWD40 and CsMYB5e expression levels in CsWD40 , CsMYB5e , and CsWD40 ♀ CsMYB5e ♂ transgenic tobacco flowers. All the data were present based on three biological and technical repeats.

Techniques Used: Transgenic Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p
Figure Legend Snippet: Effects of different abiotic treatments on the expression levels of CsWD40 in tea leaves. ( A ) The transcript levels of CsWD40 under mannitol (MAN), sodium chloride (NaCl), salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), and sucrose (Suc) stresses. ( B ) The transcript levels of CsWD40 under different temperatures. The asterisks indicate the significant level ( n = 3, * p

Techniques Used: Expressing

Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).
Figure Legend Snippet: Identification of the CsWD40 function in transgenic tobacco. ( A ) Analysis of CsWD40 transcription levels in the flowers by semiquantitative PCR. ( B ) Relative content of anthocyanin in the flowers of CsWD40 overexpressing tobacco. ( C ) Relative soluble proanthocyanidin content in the flowers of CsWD40 overexpressing tobacco. All data are the means of three biological replicates, and the error bars represent the standard deviation of three replicates. Statistical significance was analyzed using ANOVA software (ANOVA ALL MAC VERSION 2.0, Thomas Hanson, OR, USA). Means followed by the same letter are not significantly different ( p > 0.05).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Standard Deviation, Software

CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.
Figure Legend Snippet: CsWD40 complements the phenotypes of Arabidopsis ttg1 mutant. ( A ) Leaf trichome occurrence. Bar = 0.5mm ( B ) Seed coat pigmentation. ( C ) Trichome seedling.

Techniques Used: Mutagenesis

Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.
Figure Legend Snippet: Protein interactions between CsWD40 and other transcript factors from tea plants by two-hybrid system. ( A ) A model pattern of the MYB–bHLH–WD40 ternary complex. ( B ) Protein–protein interactions between CsWD40 and bHLH transcript factors (CsTT8 and CsGL3) from tea plants by two-hybrid system. ( C ) Protein–protein interactions between Cs WD40 and MYB transcript factors (CsAN2 and CsMYB5e) from tea plants by two-hybrid system.

Techniques Used:

Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.
Figure Legend Snippet: Expression of genes involved in flavonoid biosynthesis in the flowers of CsWD40-overexpressing tobacco plants. ( A ) A schematic diagram of flavonoid biosynthetic pathway. ( B ) Expression profiles of genes in flavonoid pathway in flowers of transgenic CsWD40 tobacco lines. CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; FLS, flavonol synthase; AN2, N. tabacum Anthocyanin 2; AN1a, N. tabacum Anthocyanin 1a; AN1b, N. tabacum Anthocyanin 1b.

Techniques Used: Expressing, Transgenic Assay

23) Product Images from "Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis *Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis * S"

Article Title: Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis *Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis * S

Journal: Molecular & cellular proteomics : MCP

doi: 10.1074/mcp.M400094-MCP200

Summary of the annotated functions of the 40 differentially expressed mitochondrial proteins between NSC34 WT-SOD1 and NSC-34 G93A-SOD1 cells
Figure Legend Snippet: Summary of the annotated functions of the 40 differentially expressed mitochondrial proteins between NSC34 WT-SOD1 and NSC-34 G93A-SOD1 cells

Techniques Used:

Expression levels of human SOD1 in the NSC34 cell line model of fALS The total cell lysate of NSC34 WT-SOD1 and NSC34 G93A-SOD1 cells was resolved by 12% SDS-PAGE, transferred to a nitrocellulous membrane, and blotted with an SOD1 polyclonal antibody. The endogenous mouse SOD1 and human SOD1 are indicated by arrows . Significant levels of human WT- and G93A-SOD1 are expressed in NSC34 WT-SOD1 and NSC34 G93A-SOD1 cells, respectively.
Figure Legend Snippet: Expression levels of human SOD1 in the NSC34 cell line model of fALS The total cell lysate of NSC34 WT-SOD1 and NSC34 G93A-SOD1 cells was resolved by 12% SDS-PAGE, transferred to a nitrocellulous membrane, and blotted with an SOD1 polyclonal antibody. The endogenous mouse SOD1 and human SOD1 are indicated by arrows . Significant levels of human WT- and G93A-SOD1 are expressed in NSC34 WT-SOD1 and NSC34 G93A-SOD1 cells, respectively.

Techniques Used: Expressing, SDS Page

24) Product Images from "Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality"

Article Title: Epstein-Barr Virus BGLF4 Kinase Retards Cellular S-Phase Progression and Induces Chromosomal Abnormality

Journal: PLoS ONE

doi: 10.1371/journal.pone.0039217

The BGLF4 expression level is the key factor that determines cell fates. (A) Slide-cultured GFP-H2B-HeLa cells were transfected with DsRed-BGLF4, DsRed-K102I and DsRed-monomer. At 24 h post transfection, cells were fixed with 4% paraformaldehyde, stained for DNA using Hoechst 33258 and examined using fluorescence microscopy. (B)(C)(D) GFP-H2B-HeLa cells were seeded and transfected with (B) DsRed-monomer vector or (C)(D) Ds-Red-BGLF4. At 6 h post transfection, fields of view were selected randomly and filmed using an Axiovert 200 M inverted fluorescence microscope every 10 min.
Figure Legend Snippet: The BGLF4 expression level is the key factor that determines cell fates. (A) Slide-cultured GFP-H2B-HeLa cells were transfected with DsRed-BGLF4, DsRed-K102I and DsRed-monomer. At 24 h post transfection, cells were fixed with 4% paraformaldehyde, stained for DNA using Hoechst 33258 and examined using fluorescence microscopy. (B)(C)(D) GFP-H2B-HeLa cells were seeded and transfected with (B) DsRed-monomer vector or (C)(D) Ds-Red-BGLF4. At 6 h post transfection, fields of view were selected randomly and filmed using an Axiovert 200 M inverted fluorescence microscope every 10 min.

Techniques Used: Expressing, Cell Culture, Transfection, Staining, Fluorescence, Microscopy, Plasmid Preparation

Expression of BGLF4 elicits abnormal chromosomal structure and micronuclei formation in various cell types. (A) Slide-cultured HeLa cells with stable GFP-H2B expression were transfected with DsRed-BGLF4, DsRed-K102I or pDsRed-monomer. At 48 h or 72 h post transfection, cells were fixed, stained for DNA with Hoechst 33258 and examined using fluorescence microscopy. (B) Slide-cultured 293 T-REx B22, K9, and V4 cells were treated with 100 ng/ml doxycycline (Dox). At 72 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for DNA with Hoechst 33258. Micronucleus (MN) formation was observed using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,000 cells. Bars represent mean values in MN% (duplicates±SD). One of the two independent experiments conducted is shown. (C) Slide-cultured NPC-TW01 T-REx KIT2 and VIT7 cells were incubated with 50 ng/ml Dox for 72 h. Cells were fixed with 4% paraformaldehyde and examined using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,200-1,600 cells. An asterisk (*) indicates a statistically significant difference from vector control cells (P
Figure Legend Snippet: Expression of BGLF4 elicits abnormal chromosomal structure and micronuclei formation in various cell types. (A) Slide-cultured HeLa cells with stable GFP-H2B expression were transfected with DsRed-BGLF4, DsRed-K102I or pDsRed-monomer. At 48 h or 72 h post transfection, cells were fixed, stained for DNA with Hoechst 33258 and examined using fluorescence microscopy. (B) Slide-cultured 293 T-REx B22, K9, and V4 cells were treated with 100 ng/ml doxycycline (Dox). At 72 h post induction (hpi), cells were fixed with 4% paraformaldehyde and stained for DNA with Hoechst 33258. Micronucleus (MN) formation was observed using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,000 cells. Bars represent mean values in MN% (duplicates±SD). One of the two independent experiments conducted is shown. (C) Slide-cultured NPC-TW01 T-REx KIT2 and VIT7 cells were incubated with 50 ng/ml Dox for 72 h. Cells were fixed with 4% paraformaldehyde and examined using fluorescence microscopy. Detection of MN formation (MN%) was calculated as the percentage of cells containing micronuclei, derived from the analysis of 1,200-1,600 cells. An asterisk (*) indicates a statistically significant difference from vector control cells (P

Techniques Used: Expressing, Cell Culture, Transfection, Staining, Fluorescence, Microscopy, Derivative Assay, Incubation, Plasmid Preparation

25) Product Images from "Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival"

Article Title: Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival

Journal: Infection and Immunity

doi: 10.1128/IAI.00154-12

Confirmation of positive interactions in yeast by cotransformation. Y2HGold yeast cells were cotransformed with the bait plasmid pGBKT7-TRP32C and one of the prey plasmids pGADT7-EF1A1, -DAZAP2, -CD63, -FTL, -CD14, and -PSMB1. As a control, pGBKT7 (empty vector) was used to cotransform with each prey plasmid. The positive interactions were confirmed by selection on QDO/X/A plates.
Figure Legend Snippet: Confirmation of positive interactions in yeast by cotransformation. Y2HGold yeast cells were cotransformed with the bait plasmid pGBKT7-TRP32C and one of the prey plasmids pGADT7-EF1A1, -DAZAP2, -CD63, -FTL, -CD14, and -PSMB1. As a control, pGBKT7 (empty vector) was used to cotransform with each prey plasmid. The positive interactions were confirmed by selection on QDO/X/A plates.

Techniques Used: Plasmid Preparation, Selection

26) Product Images from "Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival"

Article Title: Ehrlichia chaffeensis TRP32 Interacts with Host Cell Targets That Influence Intracellular Survival

Journal: Infection and Immunity

doi: 10.1128/IAI.00154-12

TRP32 interactions with host EF1A1, DAZAP2, CD63, and FTL are involved in E. chaffeensis infection. THP-1 cells were transfected with EF1A1, DAZAP2, CD63, FTL, or control siRNA and then infected with E. chaffeensis . (A and B) At 2 days p.i., RT-PCR and Western blotting were performed to compare the amounts of mRNA (A) and protein (B), respectively. (C) Bacterial numbers were determined by qPCR at 1, 2, and 3 days p.i. Results are from three independent experiments, and the values are means ± standard deviations (*, P
Figure Legend Snippet: TRP32 interactions with host EF1A1, DAZAP2, CD63, and FTL are involved in E. chaffeensis infection. THP-1 cells were transfected with EF1A1, DAZAP2, CD63, FTL, or control siRNA and then infected with E. chaffeensis . (A and B) At 2 days p.i., RT-PCR and Western blotting were performed to compare the amounts of mRNA (A) and protein (B), respectively. (C) Bacterial numbers were determined by qPCR at 1, 2, and 3 days p.i. Results are from three independent experiments, and the values are means ± standard deviations (*, P

Techniques Used: Infection, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction

27) Product Images from "GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product, Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein ▿"

Article Title: GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product, Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00757-09

Mapping the binding domain of KS-Bcl-2 to PICT-1. (A) Schematic representations of wild-type KS-Bcl-2 and its deletion mutants used in the mapping experiments; the boundary amino acids are numbered. The locations of the BH-1 to -4 motifs and the transmembrane
Figure Legend Snippet: Mapping the binding domain of KS-Bcl-2 to PICT-1. (A) Schematic representations of wild-type KS-Bcl-2 and its deletion mutants used in the mapping experiments; the boundary amino acids are numbered. The locations of the BH-1 to -4 motifs and the transmembrane

Techniques Used: Binding Assay

Mapping the nucleolar localization signal of PICT-1 and the requirements for KS-Bcl-2 interaction. (A) Schematic representations of wild-type PICT-1 and its deletion mutants used in the mapping experiments; the boundary amino acids are numbered. The results
Figure Legend Snippet: Mapping the nucleolar localization signal of PICT-1 and the requirements for KS-Bcl-2 interaction. (A) Schematic representations of wild-type PICT-1 and its deletion mutants used in the mapping experiments; the boundary amino acids are numbered. The results

Techniques Used:

Cellular localization of KS-Bcl-2 and PICT-1. 293T cells were transfected with the indicated expression plasmids. Mitochondrial localization of KS-Bcl-2 was confirmed with MitoTracker. Ectopically expressed myc-PICT-1 was detected by anti-myc, while endogenous
Figure Legend Snippet: Cellular localization of KS-Bcl-2 and PICT-1. 293T cells were transfected with the indicated expression plasmids. Mitochondrial localization of KS-Bcl-2 was confirmed with MitoTracker. Ectopically expressed myc-PICT-1 was detected by anti-myc, while endogenous

Techniques Used: Transfection, Expressing

Identification of PICT-1 as a KS-Bcl-2 target of interaction in yeast.
Figure Legend Snippet: Identification of PICT-1 as a KS-Bcl-2 target of interaction in yeast.

Techniques Used:

KS-Bcl-2 physically associates with PICT-1 in 293T cells. (A) To detect the association between PICT-1 and KS-Bcl-2, expression vectors containing HA-KS-Bcl-2 and myc-PICT-1 were transfected into 293T cells. Cells transfected with the HA-KS-Bcl-2 expression
Figure Legend Snippet: KS-Bcl-2 physically associates with PICT-1 in 293T cells. (A) To detect the association between PICT-1 and KS-Bcl-2, expression vectors containing HA-KS-Bcl-2 and myc-PICT-1 were transfected into 293T cells. Cells transfected with the HA-KS-Bcl-2 expression

Techniques Used: Expressing, Transfection

KS-Bcl-2 accumulates in the nucleoli when coexpressed with PICT-1. (A) 293T cells were transfected with the indicated expression plasmids. Overexpression of PICT-1 increased the nucleolar localization of KS-Bcl-2 but failed to affect the cellular localization
Figure Legend Snippet: KS-Bcl-2 accumulates in the nucleoli when coexpressed with PICT-1. (A) 293T cells were transfected with the indicated expression plasmids. Overexpression of PICT-1 increased the nucleolar localization of KS-Bcl-2 but failed to affect the cellular localization

Techniques Used: Transfection, Expressing, Over Expression

Knockdown of endogenous PICT-1 aborogates the nucleolar localization of KS-Bcl-2. (A) pSilencer 3.1 H1-hygro plasmids encoding shRNA targeting PICT-1 or control shRNA were transfected into 293T cells, and 24 h after transfection, 100 μg/ml hygomycin
Figure Legend Snippet: Knockdown of endogenous PICT-1 aborogates the nucleolar localization of KS-Bcl-2. (A) pSilencer 3.1 H1-hygro plasmids encoding shRNA targeting PICT-1 or control shRNA were transfected into 293T cells, and 24 h after transfection, 100 μg/ml hygomycin

Techniques Used: shRNA, Transfection

PICT-1 reduces the antiapoptotic activity of KS-Bcl-2. (A) 293T cells were transfected with the indicated plasmids, together with GFP expression plasmid to mark the transfected cells. All samples received equal amounts of DNA. The viability of the cells
Figure Legend Snippet: PICT-1 reduces the antiapoptotic activity of KS-Bcl-2. (A) 293T cells were transfected with the indicated plasmids, together with GFP expression plasmid to mark the transfected cells. All samples received equal amounts of DNA. The viability of the cells

Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation

28) Product Images from "Downregulation of LINC01021 by curcumin analog Da0324 inhibits gastric cancer progression through activation of P53"

Article Title: Downregulation of LINC01021 by curcumin analog Da0324 inhibits gastric cancer progression through activation of P53

Journal: American Journal of Translational Research

doi:

Overexpression of LINC01021 reverses Da0324-mediated cytotoxic effects in gastric cancer cells. A. BGC823 and SGC7901 cells were transfected with negative control vector (pLVX-NC) or overexpressing LINC01021 vector (pLVX- LINC01021 ) for 48 h. LINC01021 expression was determined by qRT-PCR analysis. The data represent the mean ± SD from three independent experiments. ***, P
Figure Legend Snippet: Overexpression of LINC01021 reverses Da0324-mediated cytotoxic effects in gastric cancer cells. A. BGC823 and SGC7901 cells were transfected with negative control vector (pLVX-NC) or overexpressing LINC01021 vector (pLVX- LINC01021 ) for 48 h. LINC01021 expression was determined by qRT-PCR analysis. The data represent the mean ± SD from three independent experiments. ***, P

Techniques Used: Over Expression, Transfection, Negative Control, Plasmid Preparation, Expressing, Quantitative RT-PCR

Da0324 or LINC01021 -specific ASO treatment suppresses tumor growth of GC cells in vivo . A. Effects of Da0324 treatment on tumor volume in subcutaneous xenograft mouse models using KATO III cells. Tumor volume as measured by caliper of Da0324 (20 mg/kg, intraperitoneal injection every day) or vehicle administered for 18 days in a subcutaneous xenograft model. The data were expressed as mean ± SD. *, P
Figure Legend Snippet: Da0324 or LINC01021 -specific ASO treatment suppresses tumor growth of GC cells in vivo . A. Effects of Da0324 treatment on tumor volume in subcutaneous xenograft mouse models using KATO III cells. Tumor volume as measured by caliper of Da0324 (20 mg/kg, intraperitoneal injection every day) or vehicle administered for 18 days in a subcutaneous xenograft model. The data were expressed as mean ± SD. *, P

Techniques Used: Allele-specific Oligonucleotide, In Vivo, Injection

ASO-mediated silencing of LINC01021 inhibits gastric cancer cell migration, invasion, and EMT. A. BGC823 and SGC7901 cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1 or LINC01021 -specific ASO-2 for 48 h and transwell migration and matrigel invasion assays were performed. Representative image (left panel) and quantitative data (right panel) of cell migration and invasion are shown. All data are presented as the means ± SD. ***, P
Figure Legend Snippet: ASO-mediated silencing of LINC01021 inhibits gastric cancer cell migration, invasion, and EMT. A. BGC823 and SGC7901 cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1 or LINC01021 -specific ASO-2 for 48 h and transwell migration and matrigel invasion assays were performed. Representative image (left panel) and quantitative data (right panel) of cell migration and invasion are shown. All data are presented as the means ± SD. ***, P

Techniques Used: Allele-specific Oligonucleotide, Migration, Transfection

ASO-mediated silencing of LINC01021 inhibits cell proliferation and induces apoptosis and autophagy in gastric cancer cells. A. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1, or LINC01021 -specific ASO-2 for 48 h. LINC01021 expression were determined by qRT-PCR. B. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1, or LINC01021 -specific ASO-2. At 0, 24, 48 or 72 h post-transfection, cell viability was determined by the CCK-8 assays. The data were expressed as mean ± SD. All data are representative of three independent experiments. **, P
Figure Legend Snippet: ASO-mediated silencing of LINC01021 inhibits cell proliferation and induces apoptosis and autophagy in gastric cancer cells. A. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1, or LINC01021 -specific ASO-2 for 48 h. LINC01021 expression were determined by qRT-PCR. B. BGC823, SGC7901 and KATO III cells were transfected with 50 nM ASO-control, LINC01021 -specific ASO-1, or LINC01021 -specific ASO-2. At 0, 24, 48 or 72 h post-transfection, cell viability was determined by the CCK-8 assays. The data were expressed as mean ± SD. All data are representative of three independent experiments. **, P

Techniques Used: Allele-specific Oligonucleotide, Transfection, Expressing, Quantitative RT-PCR, CCK-8 Assay

Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P
Figure Legend Snippet: Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P

Techniques Used: Next-Generation Sequencing, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, Expressing

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Amplification:

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Purification:

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
Article Snippet: .. Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

Polymerase Chain Reaction:

Article Title: Capped antigenomic RNA transcript facilitates rescue of a plant rhabdovirus
Article Snippet: .. The PCR product was amplified using the primers SYNV HRz3 F/SYVV HRz3 R and assembled through an In-Fusion HD PCR Cloning kit (Clontech, Japan). .. To yield the p35S–HHm-SYNV MRGFP-RFP , the HH2 catalytic sites were mutated from GUC to GUG by mutagenesis PCR using the primers TG+ SYNV(+)1 Xma I F and T-DNA LB Spe I Pvu I R, the resulting HHm PCR products were digested with Xma I and Sac I and then inserted into the intermediate plasmid.

Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
Article Snippet: .. Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech). .. The correct sequences of wild-type (WT; pTB2ex13WT ) and mutant plasmids (pTB2ex13DEL ) were verified by DNA sequencing.

Gel Extraction:

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
Article Snippet: .. Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

Chloramphenicol Acetyltransferase Assay:

Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants
Article Snippet: .. Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions. .. Following confirmation with RFLP analysis, plasmids were verified by DNA sequencing.

Over Expression:

Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
Article Snippet: .. Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech) .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare)

Plasmid Preparation:

Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
Article Snippet: .. The Citrine responder (pJFRC81_3×VAS1 -Syn21-Citrine-HA-P10) was assembled from plasmid pJFRC81_3×VAS-1-GFP-P10 digested with BglII and EcoRI, and fragments Syn21-Citrine-HA, VAS-1-HA-P10, Mhc-SD by in-fusion cloning (Clontech, 639648). .. The Cerulean responder was assembled from plasmid 3×VAS-2-GFP-P10 digested with AatII and EcoRI, and fragments Hsp70-Syn21, FLAG-Cerulean and VAS-2-P10 by in-fusion cloning.

Article Title: Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method
Article Snippet: .. Yeast strains (see below) Yeast growth medium (YEP, SC supplemented with appropriate carbon source) Plasmid for transcription factor overexpression (see below) In-Fusion HD cloning kit (Clontech) .. 37% formaldehyde (Fisher Scientific) 2.5 M glycine (Bio-Rad) or 3 M Tris base pH 8.0 (Sigma) Benoit Extraction Buffer (200 mM Tris-HCl (pH 8.0), 400 mM (NH4 )2 SO4 , 10 mM MgCl2 , 1 mM EDTA, 10% glycerol, 7 mM β-mercaptoethanol, and protease inhibitors as described below) Protease inhibitors: Roche Complete Protease Inhibitor Cocktail Tablet OR 1.0 mM phenylmethylsulfonyl fluoride, 2.0 mM benzamidine, 2.0 µM pepstatin, 0.6 µM leupeptin, and 2.0 µg of chymostatin per ml of buffer SDS polyacrylamide gel electrophoresis and transfer system (mini-PROTEAN tetra cell, Bio-Rad) Transcription factor-specific primary antibody ECL anti-rabbit or –mouse horseradish peroxidase linked whole antibody (GE Healthcare) Amersham ECL Prime western blotting detecting reagent (GE Healthcare)

Article Title: Pullulanase Is Necessary for the Efficient Intracellular Growth of Francisella tularensis
Article Snippet: .. The amplified pulB gene was ligated into the pCold™ TF DNA plasmid using the In-Fusion HD Cloning Kit (Takara, Shiga, Japan) in accordance with the manufacturer’s instructions. .. The resulting plasmid DNA was used to transform Competent High DH5α (Toyobo, Tokyo, Japan) according to the manufacturer's instructions, and the transformed E . coli cells were subsequently spread onto LB agar plates containing 50 μg/ml ampicillin.

Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies
Article Snippet: .. The mCherry responder was assembled from plasmid pJFRC81_3×VAS-3-GFP-P10 (digested with BglII and EcoRI) and fragments Syn21-V5-mCherry, VAS-3-P10, Mhc-SA by in-fusion cloning (Clontech, 639648) resulting in pJFRC81_3×VAS3 -Syn21-V5-mCherry-P10. .. For the generation of a Citrine-Cerulean double responder vector, the 3×VAS4 -hsp70-Syn21-FLAG-Cerulean-P10 cassette was amplified from plasmid pJFRC81_3×VAS4 -Syn21-FLAG-Cerulaen-P10 using primers 3 ×VAS 4 -Cerulean-Fwd and 3 ×VAS 4 -Cerulean-Rev .

Article Title: Reccurrent F8 Intronic Deletion Found in Mild Hemophilia A Causes Alu Exonization
Article Snippet: .. Amplicons were inserted in the Nde I restriction site of the previously described pTB minigene vector using the In-fusion HD PCR cloning kit (Clontech). .. The correct sequences of wild-type (WT; pTB2ex13WT ) and mutant plasmids (pTB2ex13DEL ) were verified by DNA sequencing.

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